NAME : KHADIJAH BINTI MOHAMAD NIZAM CLASS : M12E

TITLE The number of yeast cell in primary squares that determines by the different dilution of yeast suspension AIM To investigate the effect of different dilution of yeast suspension on the number of yeast in primary squares. RESEARCH QUESTION Does the different dilution of yeast suspension affects the number of yeast in primary squares?

INTRODUCTION The hemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to determine the concentration of cells in a liquid sample. It is frequently used to determine the concentration of blood cells (hence the name hemo-) but it is also used to count the number of microscopic particles. Haemocytometer is a specialized slide that can accurately determine the number of cells in a specified volume of liquid. There are several rules to get the accurate reading. The first is the fluid containing the cells must be appropriately prepared before applying it to the hemocytometer. The fluid should be a homogenous suspension. Cells that stick together in clumps are difficult to count and they are not evenly distributed. Cells that are on the line of a grid require special attention. Cells that touch the top and right lines of a square should not be counted, cells on the bottom and left side should be counted.1

October 24, 2010. The Haemocytometer (Counting Chamber):http://www.microbehunter.com/2010/06/27/thehemocytometer-counting-chamber/ visited August 4, 2012

Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with 1,500 species currently described (estimated to be only 1% of all fungal species). Yeasts are unicellular, although some species with yeast forms may become multicellular through the formation of a string of connected budding cells known as pseudohyphae, or false hyphae, as seen in most molds.2 The factors that affecting the population of yeast are temperature, pH, type of sugar and concentration of yeast suspension3. In this experiment, we are focusing in concentration of yeast suspension. The higher the dilution of yeast suspension, the lower the density of yeast in squares. This is because as the dilution of yeast suspension higher, the concentration of yeast becomes lower. This contributes to the lower density of yeast in squares.

HYPOTHESIS If the dilution of yeast suspension is higher, then the density of yeast in primary squares also will be higher. When there is higher in dilution of yeast suspension, the yeast concentration will be lower. Thus, this causes the density of yeast in primary squares to be lower.

July 18, 2012. Yeast: http://en.wikipedia.org/wiki/Yeast. Visisted August 4, 2012

the Australian of Food Science & Technology Incorporated. Factors Affect Yeast Growth ttp://www.curriculumsupport.education.nsw.gov.au/secondary/science/assets/aifst/Experiments/Yeast%20growth.pd f. visited August 4, 2012

VARIABLES

Independent variable

Concentration of yeast suspension used which are 10x, 100x, 1000x, 10000x, 100000x

Dependent variable

Population of yeast cell that seen under the microscope is counted by using haemocytometer

Constant variables

1. Number of grid used to count the number of yeast population Use 4 grids of haemocytometer to count the number of yeast population.

2. Temperature of yeast suspension Temperature of yeast suspension is fixed at room temperature.

3. Types of diluent Diluent used in this experiment is fixed, that is distilled water.

4. Volume of diluent Distilled water is fixed at 9 ml to be add in each test tube containing yeast suspension.

APPARATUS & MATERIALS APPARATUS 10 ml Measuring cylinder 50 ml beaker Test tube Test tube rack Petri dish haemocytometer QUANTITY 2 1 6 1 1 1

MATERIALS Yeast suspension Ethanol Distilled water Tissue paper

QUANTITY 1ml 2 drops 60 ml 5 sheets

PROCEDURES A. SETTING UP THE SLIDE

1. Haemocytometer is cleaned with ethanol, and then wiped with lens tissue. 2. The slide is moistening using the damp tissue as shown in diagram 1

Diagram 1

3. The special coverslip is pushed on the slide as shown in diagram, the outside edges of the coverslip is pressed down at the same time until the Newton`s rings can be seen (Diagram 2)

Diagram 2

B. LOADING THE HAEMOCYTOMETER

4. Yeast cell is shaken gently. 5. The end of the capillary tube is inserted into the suspension. The liquid will rise into the tube.

6. The end of the capillary tube is run along the edge of the coverslip between the arms of the `H`. The area between the coverslip and the top half of the `H` is filled with suspension. (Diagram 3)

Diagram 3

7. The slide is turn 180o and the process is repeated for the opposite edge of the coverslip. (diagram 4)

Diagram 4

8. The haemocytometer is placed on a damp tissue in a petri dish for at least 2 minutes to equilibrate.

C. Counting the cells

The haemocytometer has two grids situated as shown in diagram 5:

Diagram 5

9. Haemocytometer is placed on the microscope stage. 10. The grid lines of the haemocytometer are focused using the 10x objective of the microscope. One set of corner/primary square is focused. It is indicated by the circle in diagram 6.

Diagram 6 : haemocytometer diagram indicating the corner / primary square which should be used for counting.

Diagram 6

11. The number of cells in the area of primary Square is counted using hand tally. The cells are count if they are within the primary square and any positioned on the top or the right side of the square (Diagram 7)

Diagram 7

12. Steps 1-12 are repeated using 5 series of dilution.

QUANTITATIVE DATA DILUTION OF YEAST SUSPENSION SQUARE 1 SQUARE 2 SQUARE 3 SQUARE 4 TOTAL NUMBER OF YEAST CELLS IN EACH SQUARES ( 4 CELLS)

111 21 56 8 2

59 16 90 5 3

164 19 48 11 6

62 22 49 6 1

394 243 78 30 12

Table 1: shows the different dilution of yeast suspension that affects number and the total number of yeast cells in each square. *dilution is data from our group. The rest of the data is a pooled data.

QUALITATIVE DATA 1. The color if yeast suspension is light yellow. 2. As the dilution higher, the color of the yeast suspension goes clearer and lastly when the dilution is 1000000X, the color becomes slightly transparent.

DATA PROCESSING 1. The volume of yeast cell that can occupy the one primary square is 0.1 mm3 (1.0 mm2 x 0.1 mm) Area of one primary square Depth of primary square Volume of primary square = 1.0 mm2 = 0.1 mm = 1.0 mm2 x 0.1mm = 0.1 mm3 2. Number of yeast cells in 1 cm3 =n x 2.5 x 1000

Formula for calculating the density of yeast cell Density =

number of yeast cell in

3. Sample calculation of density of yeast cell for the dilution 10X

Density= = 2475000

PRESENTATION OF DATA PROCESSING

DILUTION OF YEAST SUSPENSION

NUMBER OF YEAST CELLS IN EACH SQUARES ( 4 CELLS) SQUARE 1 SQUARE 2 SQUARE 3 SQUARE 4 TOTAL

DENSITY OF YEAST CELL

111 21 56 8 2

59 16 90 5 3

164 19 48 11 6

62 22 49 6 1

394 243 78 30 12

2475000 1518750 195000 75000 30000

Table 2 shows the number of yeast cells in each squares and density of yeast cells when using different dilution of yeast suspension.

DISCUSSION From the result, we can say that as the dilution higher, the number of yeast cell will be lower. As a result, the density of yeast cell will be lower too. This is because as the dilution higher, the diluent will be higher than the yeast suspension in the mixture. This causes the yeast cell to be diffused between the diluent and break apart. Thus, this causing the density of yeast cell to be lower in the high dilution mixture rather than the lower one. CONCLUSION The higher the dilution of yeast suspension, the lower the density of the yeast cell in primary square. As the dilution of yeast suspension is higher, the number of cells will be lower. Hypothesis is accepted.

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EVALUATION LIMITATIONS AND WEAKNESSES 1. The yeast cell and other small microorganisms are difficult to distinguish as under the microscope, they are look alike. 2. The grids of haemocytometer are difficult to be seen under the microscope as they are too small. SUGGESTIONS 1. Before observing them under the microscope, the dilution is done to get clearer observation of yeast cells. 2. The magnification of microscope is adjust until the clearer grids of haemocytometer is seen.

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